Nonhuman pregnane x receptor sequences for use in comparative pharmacology

ABSTRACT

Polynucleotides and polypeptides of canine, primate, porcine and fish pregnane X receptor (PXR), as well as expression vectors and host cells for expression of these PXR receptors, are provided. Also provided are methods for screening for modulators of these PXR receptors and using these receptors for comparative pharmacology and in selection of appropriate preclinical animal models predictive of human PXR activity.

FIELD OF THE INVENTION

[0001] The present invention relates to nonhuman pregnane X receptor(PXR) polynucleotide and polypeptide sequences isolated from fish,canines, porcine and primates and their use in comparative pharmacology.

BACKGROUND OF THE INVENTION

[0002] Members of the cytochrome P450 family of hemoproteins arecritical in the oxidative metabolism of a wide variety of endogenoussubstances and xenobiotics, including various carcinogens and toxins(Nebert et al. (1987) Ann. Rev. Biochem. 56:945-993). In man, P450 3A4monooxygenase, also referred to as CYP3A4 monooxygenase, plays a majorrole in the biotransformation of drugs due to its abundance in liver andintestine and its broad substrate specificity. P450 3A4 catalyzes themetabolism of >60% of all drugs that are in use including steroids,immunosuppressive agents imidazole antimycotics, and macrolideantibiotics (Maurel, P. in Cytochrome P450: metabolic and toxicologicalaspects (ed. Ioannides, C.) 241-270 (CRC Press, Inc. Boca Raton, Fla.1996).

[0003] Expression of. P450 3A4 is induced both in vivo and in primaryhepatocytes in response to treatment with a variety of compoundsincluding, but not limited to, commonly used drugs such as theglucocorticoid dexamethasone, the antibiotic rifampicin, the antimycoticclotrimazole, and the hypocholesterolemic agent lovestatin (Maurel, P.in Cytochrome P450: metabolic and toxicological aspects (ed. Ioannides,C.) 241-270 (CRC Press, Inc. Boca Raton, Fla. 1996); Guzelian, P. S. inMicrosomes and Drug Oxidation (eds. Miners, J. O., Birkett, D. J., Drew,R. & McManus, M.) 148-155 (Taylor and Francis, London, 1988)). Theinducibility of P450 3A4 expression levels coupled with its broadsubstrate specificity represent the basis for many drug interactions inpatients undergoing combination therapy. Thus, analysis of the effectsof new compounds on P450 3A4 gene expression is an important aspect indrug development.

[0004] While attempts have been made to develop in vivo and in vitroassays with which to profile the effects of compounds on P450 3A4expression levels, poorly understood species-specific variations havelimited the utility of using animals and their tissues for testingpurposes. Thus, analysis of the effects of new compounds on P450 3A4gene expression has been largely restricted to laborious assaysinvolving human liver tissue.

[0005] Nuclear hormone receptors comprise a large superfamily ofligand-modulated transcription factors that, in part, mediate responsesto steroids, retinoids, and thyroid hormones (for review see Beato etal., (1995) Cell 83:851-857; Kastner et al., (1995) Cell 83:859-869;Mangelsdorf and Evans, (1995) Cell 83:841-850). Detailed analysis of thereceptors, most notably the steroid class of receptors, has revealedmultiple discrete functional modules within the family that displaygeneralized functional characteristics (Tzukerman et al., (1994) Mol.Endocrinol. 8:21-30). A variable amino-terminal domain (A/B) is presentthat typically contains a strong and autonomous activation function(AF1), shown to be critical for cell and target gene specificity (Toraet al., (1988) Nature 333: 677-684). A more carboxyl-terminal centralregion contains a DNA binding domain (DBD) characterized by two C4-typezinc fingers. The DBD binds to specific genomic response elements andthereby regulates the transcriptional activity of select genescontaining the response elements. At the distal carboxyl terminus, aligand binding domain (LBD) is present containing a highly conservedsecond transactivation function (AF2) that is important forhormone-dependent transcriptional transactivation (Lanz and Rusconi,(1994) Endocrinology 135: 2183-2195). Sequences that function in nuclearlocalization, receptor dimerization, and interaction with heat-shockproteins (Gronemeyer and Laudet, (1995) CCQ 2:1173-1308) are alsopresent within the nuclear receptor substructure. Through thecoordinated action of these separate functional domains, nuclearreceptor activation by ligand culminates in modulation of target geneexpression (Tsai and O'Malley, (1994) Ann. Rev. Biochem. 63: 451-486)and in certain cases, cross-talks with other cell signaling pathwayssuch as the NF-kB (Stein and Yang, (1995) Mol. Cell. Biol. 15:4971-4979) and AP-1 (Paech et al., (1998) Science 277: 1508-1510).Ultimately, ligand alters nuclear receptor function by altering theconstellation of protein-protein interactions in which the receptor isengaged (for review, see Freedman, (1999) Cell 97: 5-8). The moleculardetails underlying the multitude of cellular effects mediated by nuclearreceptors are the subject of intense research activity.

[0006] For the nuclear receptor for pregnane X, referred to herein asPXR (Unified Nomenclature Committee designation NR1I2), it has beenshown that PXR cell-based and binding assays are predictive of in vivoeffects on the cytochrome P450 3A4 gene (Kliewer et al. (1998) Cell 92,73-8273-82;

[0007] Lehmann et al. (1998) J. Clin. Invest. 102, 1016-1023; Jones etal. (2000) Mol. Endo. 27-39).

[0008] WO 99/48915 discloses human PXR which binds to the CYP promoterrifampicin/dexamethasone response element in cytochrome P450 3A4. Alsodisclosed are nucleic acid sequences encoding human PXR, as well asvectors and host cells for expression of the human receptor, and methodsfor using this receptor in vitro to screen compounds for their abilityto modulate P450 3A4 expression in humans.

[0009] However, nonhuman animal PXRs, particularly animals well acceptedfor use in preclinical studies, would also be useful in the developmentin vitro and in vivo animal models 35 for profiling the effects ofcompounds on P450 3A4 expression levels and to select preclinical modelspredictive of effects in humans. Additionally, dissecting the broaderbiological hysiological role of PXR beyond cytochrome P450 geneinduction is facilitated by an understanding of which compounds activatePXR in animal models of interest. Thus, comparative pharmacology of PXRopens the possibility of extending the utility of PXR in drugdevelopment and toxicity assays as well as validating this receptor as auseful target in disease.

[0010] Mouse PXR has been cloned and sequenced (Kliewer et al. (1998)Cell 92:73-82). Rat and rabbit PXR have also been cloned and sequenced(Jones et al. (2000) Molecular Endocrinology 14(l):27-39; Zhang et al.(1999) Archives of Biochemistry and Biophysics 368(1):14-22).

[0011] The present invention relates to PXRs for other nonhuman animals.

SUMMARY OF THE INVENTION

[0012] Polynucleotide and polypeptide sequences for pregnane X receptors(PXR) isolated from fish, canines, porcine and primates are provided.The novel PXR sequences are useful as screening targets for theidentification and development of selective PXR compounds. These agentsare particularly useful in PXR comparative pharmacology and selectingappropriate animal models for preclinical studies predictive of effectsin humans.

[0013] Accordingly, the present invention provides isolated PXRpolypeptides comprising:

[0014] (a) an amino acid sequence of SEQ ID NO: 2, 4, 6 or 8;

[0015] (b) a variant of an amino acid sequence as defined in (a) whichmodulates P450 3A4 levels or activity; or

[0016] (c) a fragment of (a) or (b) which modulates P450 3A4 levels oractivity.

[0017] According to another aspect of the invention there is providedpolynucleotides encoding polypeptides of the invention, saidpolynucleotides comprising:

[0018] (a) a nucleic acid sequence of SEQ ID NO: 1, 3, 5, or 7;

[0019] (b) a nucleic acid sequence which hybridizes under stringentconditions to the nucleic acid sequence as defined in (a);

[0020] (c) a nucleic acid sequence that is degenerate as a result of thegenetic code to the nucleic acid sequences as defined in (a) or (b); or

[0021] (d) a nucleic acid sequence having at least 60% identity to thenucleic acid sequences as defined in (a), (b) or (c).

[0022] Other related aspects of the present invention include expressionvectors comprising polynucleotides of the invention which are capable ofexpressing a polypeptide of the invention, host cells comprising anexpression vector of the invention, methods of producing a polypeptideof the invention which comprise maintaining a host cell of the inventionunder conditions suitable for obtaining expression of the polypeptideand isolating said polypeptide, antibodies specific for a polypeptide ofthe invention, and transgenic nonhuman animals expressing a mutant PXRor a PXR from another species.

[0023] The present invention also provides methods for identification ofsubstances that modulate PXR activity and/or expression. In oneembodiment, the method comprises contacting a polypeptide,polynucleotide, expression vector or host cell of the invention with atest substance and determining the effect of the test substance on theactivity and/or expression of the polypeptide to determine whether thetest substance modulates PXR activity and/or expression. In anotherembodiment, the test substance is administered to a nonhuman transgenicanimal expressing a mutant PXR or a PXR from a different species and theeffects of the test substance on expression and/or activity of thereceptor are examined.

[0024] In addition, the present invention relates to compounds whichmodulate PXR activity and which are identifiable by the methods referredto above.

BRIEF DESCRIPTION OF THE SEQUENCES

[0025] SEQ ID NO: 1 shows a nucleotide and amino acid sequence of aligand binding domain of a canine pregnane X receptor.

[0026] SEQ ID NO: 2 shows the amino acid sequence of the ligand bindingdomain of the canine pregnane X receptor as depicted in SEQ ID NO:1.

[0027] SEQ ID NO:3 shows a nucleotide and amino acid sequence of aprimate pregnane X receptor.

[0028] SEQ ID NO:4 shows the amino acid sequence of the primate pregnaneX receptor as depicted in SEQ ID NO:3.

[0029] SEQ ID NO:5 shows a nucleotide and amino acid sequence of aligand binding domain of a porcine pregnane X receptor.

[0030] SEQ ID NO:6 shows the amino acid sequence of the ligand bindingdomain of the porcine pregnane X receptor as depicted in SEQ ID NO:5.

[0031] SEQ ID NO:7 shows a nucleotide and amino acid sequence of aligand binding domain of a Zebrafish pregnane X receptor.

[0032] SEQ ID NO:8 shows the amino acid sequence of the ligand bindingdomain of the Zebrafish pregnane X receptor as depicted in SEQ ID NO:7.

BRIEF DESCRIPTION OF THE FIGURES

[0033]FIG. 1 is a bargraph showing the activation of a primate PXR inthe presence of various steroids and xenobiotics.

[0034]FIG. 2 is a bargraph showing the activation of a canine PXR in thepresence of various steroids and xenobiotics.

[0035]FIG. 3 is a bargraph showing the activation of a porcine PXR inthe presence of various steroids and xenobiotics.

[0036]FIG. 4 is a bargraph showing the activation of a fish PXR in thepresence of various steroids and xenobiotics.

[0037]FIG. 5 is a bargraph showing the activation of a primate PXR inthe presence of various bile acids.

[0038]FIG. 6 is a bargraph showing the activation of a canine PXR in thepresence of various bile acids.

[0039]FIG. 7 is a bargraph showing the activation of a porcine PXR inthe presence of various bile acids.

[0040]FIG. 8 is a bargraph showing the activation of a fish PXR in thepresence of various bile acids.

DETAILED DESCRIPTION OF THE INVENTION

[0041] Throughout the present specification and the accompanying claimsthe words “comprise” and “include” and variations such as “comprises”,“comprising”, “includes” and “including” are to be interpretedinclusively. That is, these words are intended to convey the possibleinclusion of other elements or integers not specifically recited, wherethe context allows.

[0042] The present invention relates to nonhuman animal orthologs ofpregnane X receptors, referred to herein as PXRs, and variants thereof.More specifically, the present invention relates to PXRs isolated from acanine, porcine, primate or fish. Nucleotide sequence information forthe full length monkey PXR of the present invention is provided in SEQID NO: 3. A polypeptide sequence of the monkey PXR is also provided inSEQ ID NO: 4. Sequence information for the ligand binding domains of thedog, pig and Zebrafish PXRs of the present invention is provided in SEQID NO: 1, 5 and 7 (nucleotide and amino acid), respectively.Polypeptides of the ligand binding domains of the dog, pig and ZebrafishPXRs are also provided in SEQ ID NO: 2, 6, and 8.

[0043] Polypeptides of the invention consist essentially of the aminoacid sequences of SEQ ID NO: 2, 4, 6 or 8, a variant of that sequence,or a fragment of either thereof. Polypeptides of the invention may be ina substantially isolated form. It will be understood that thepolypeptide may be mixed with carriers or diluents which will notinterfere with the intended purpose of the polypeptide and still beregarded as substantially isolated. A polypeptide of the invention mayalso be in a substantially purified form, in which case it willgenerally comprise the polypeptide in a preparation in which more than50%, e.g. more than 80%, 90%, 95% or 99%, by weight of the polypeptidein the preparation is a polypeptide of the invention. Routine methods,can be employed to purify and/or synthesize the polypeptides accordingto the invention. Such methods are well understood by persons skilled inthe art, and include techniques such as those disclosed in Sambrook etal, Molecular Cloning: a Laboratory Manual, 2^(nd) Edition, CSHLaboratory Press, 1989, the disclosure of which is included herein inits entirety by way of reference.

[0044] The term “variant” refers to a polypeptide that has a sameessential character or basic biological functionality as the selectedPXR. As demonstrated herein, biological functionalities of nonhuman PXRsvary between species. By “selected PXR receptor”, as used herein, it ismeant a canine, primate, porcine or fish PXR as described herein.Further, for purposes of this invention a variant polypeptide ispreferably one which binds to the same ligand as one or more of thenonhuman animal PXRs described herein. Preferably the polypeptidemodulates P450 3A4 expression in primates, canines, porcine and/or fish.A polypeptide having a same essential character as a selected PXR of thepresent invention can be identified by monitoring for activation of theselected PXR by an inducer of P450 3A4. A full-length variantpolypeptide is preferably one which includes the entire ligand bindingdomain of the selected PXR.

[0045] In another aspect of the invention, a variant is one which doesnot show the same activity as the selected PXR but rather inhibits abasic function of the PXR. For example, a variant polypeptide is onewhich inhibits activation of a selected PXR upon exposure to an inducerof P450 3A4, for example by binding to a PXR ligand to prevent activitymediated by ligand binding to the selected PXR.

[0046] Typically, polypeptides with more than about 65% identitypreferably at least 80% , at least 85% or at least 90% and particularlypreferably at least 95%, at least 97% or at least 99% identity, with theamino acid sequences of SEQ ID NO: 2, 4, 6 or 8 are considered asvariants of the proteins. Identity can be determined using a programsuch as a BLAST sequence alignment program. Such variants may includeallelic variants and the deletion, modification or addition of singleamino acids or groups of amino acids within the protein sequence, aslong as the peptide maintains a basic biological functionality of theselected PXR.

[0047] Amino acid substitutions may be made, for example from 1, 2 or 3to 10, 15, 20 or 30 substitutions. The modified polypeptide generallyretains the same activity as a selected PXR. Conservative substitutionsmay be made, for example according to the following Table 1. Amino acidsin the same block in the second column and preferably in the same linein the third column may be substituted for each other. TABLE 1 ALIPHATICNon-polar G A P I L V polar-uncharged C S T M N Q Polar-charged D E K RAROMATIC H F W Y

[0048] Exemplary substitutions are shown in Table 2. TABLE 2 OriginalResidue Exemplary Substitutions Ala Gly; Ser Arg Lys Asn Gln; His AspGlu Cys Ser Gln Asn Glu Asp Gly Ala His Asn; Gln Ile Leu; Val Leu Ile;Val Lys Arg Met Met; Leu; Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp; Phe ValIle; Leu

[0049] Shorter polypeptide sequences, also referred to herein as“fragments” are within the scope of the invention. For example, apeptide of at least 15, 20 or 30 amino acids or up to 50, 60, 70, 80,100, 150 or 200 amino acids in length is considered to fall within thescope of the invention as long as it demonstrates a basic biologicalfunctionality of a selected PXR. In particular, but not exclusively,this aspect of the invention encompasses the situation when the proteinis a fragment of the complete protein sequence and may representparticularly, the A/B domain (e.g., about amino acids 1-40 of SEQ IDNO:4), the DBD (e.g., about amino acids 41-105 of SEQ ID NO:4) or theLBD (e.g., about amino acids 106-434 of SEQ ID NO:4 or SEQ ID NO:2, 6 or8), alone or in combination. Such fragments can be used to constructchimeric receptors preferably with another nuclear receptor, morepreferably with another member of the family of nuclear receptorsinvolved in P450 3A4 regulation such as the CAR nuclear receptor, or asintermediates in the production of the full length sequences.

[0050] Such fragments of PXR or a variant thereof can also be used toraise anti-PXR antibodies. In this embodiment, the fragment may comprisean epitope of a selected PXR polypeptide and may otherwise notdemonstrate the ligand binding or other properties of the selected PXR.

[0051] Polypeptides of the invention may be chemically modified, e.g.post-translationally modified. For example, they may be glycosylated orcomprise modified amino acid residues. They may also be modified by theaddition of histidine residues to assist their purification or by theaddition of a signal sequence to promote insertion into the cellmembrane. Such modified polypeptides fall within the scope of the term“polypeptide” of the invention.

[0052] The invention also includes nucleotide sequences that encode forcanine, primate, porcine or fish PXR or a variant thereof, as well asnucleotide sequences which are complementary thereto. The nucleotidesequence may be RNA or DNA including genomic DNA, synthetic DNA or cDNA.Preferably the nucleotide sequence is a DNA sequence, and mostpreferably a cDNA sequence. Nucleotide sequence information for thecanine, primate, porcine and Zebrafish PXRs of the present invention areprovided in SEQ ID NO: 1, 3, 5 and 7, respectively. Such nucleotides canbe isolated from cells of the selected species, namely, canine, primate,porcine or Zebrafish, or synthesized according to methods well known inthe art, as described by way of example in Sambrook et al, 1989.

[0053] Typically a polynucleotide of the invention comprises acontiguous sequence of nucleotides which is capable of hybridizing underselective conditions to the coding sequence or the complement of thecoding sequence of either SEQ ID NO: 1, 3, 5 or 7.

[0054] The polynucleotide can be in single-, double- or triple-strandedform. In addition, polynucleotide, as used herein, can refer totriple-stranded regions comprising RNA or DNA or both. The strands insuch regions may be from the same molecule or from different molecules.As used herein the term polynucleotide includes nucleic acids thatcontain one or more modified (e.g., tritylated) or unusual (e.g.,inosine) bases. Thus, DNAs or RNAs with backbones modified for stabilityor for other reasons are polynucleotides as that term is intendedherein.

[0055] A polynucleotide of the invention can hybridize to the codingsequence or the complement of the coding sequence of SEQ ID NO: 1, 3, 5or 7 at a level significantly above background. Background hybridizationmay occur, for example, because of other cDNAs present in a cDNAlibrary. The signal level generated by the interaction between apolynucleotide of the invention and the coding sequence or complement ofthe coding sequence of SEQ ID NO: 1, 3, 5 or 7 is typically at least10-fold, preferably at least 100-fold, as intense as interactionsbetween other polynucleotides and the coding sequence of SEQ ID NO: 1,3, 5 or 7. The intensity of interaction may be measured, for example, byradiolabeling the probe, e.g. with ³²P. Selective hybridization maytypically be achieved using conditions of medium to high stringency.However, such hybridization may be carried out under any suitableconditions known in the art (see Sambrook et al, 1989). For example, ifhigh stringency is required suitable conditions include from 0.1 to0.2×SSC at 60° C. up to 65° C. If lower stringency is required suitableconditions include 2×SSC at 60° C.

[0056] The coding sequence of SEQ ID NO: 1, 3, 5 or 7 may be modified bynucleotide substitutions, for example from 1, 2 or 3 to 10, 15, 25, 50or 100 substitutions. The polynucleotide of SEQ ID NO: 1, 3, 5 or 7 mayalternatively or additionally be modified by one or more insertionsand/or deletions and/or by an extension at either or both ends. Apolynucleotide may include one or more introns. For example, in oneembodiment, the polynucleotide may comprise genomic DNA. Additionalsequences such as signal sequences which may assist in insertion of thepolypeptide in a cell membrane may also be included. The modifiedpolynucleotide generally encodes a polypeptide which has the sameactivity as a selected PXR. Alternatively, a polynucleotide encodes aligand-binding portion of a polypeptide or a polypeptide which inhibitsan activity of a selected PXR. Degenerate substitutions may be madeand/or substitutions may be made which would result in a conservativeamino acid substitution when the modified sequence is translated, forexample as shown in Table 1 or 2 above.

[0057] A nucleotide sequence which is capable of selectively hybridizingto the complement of the DNA coding sequence of SEQ ID NO: 1, 3, 5, or 7will generally have at least 60%, at least 70%, at least 80%, at least90%, at least 95%, at least 98% or at least 99% sequence identity to thecoding sequence of SEQ ID NO: 1, 3, 5 or 7 over a region of at least 20,preferably at least 30, for instance at least 40, at least 60, morepreferably at least 100 contiguous nucleotides, or most preferably overthe full length of SEQ ID NO: 1, 3, 5 or 7.

[0058] For example the UWGCG Package provides the BESTFIT program whichcan be used to calculate homology (for example used on its defaultsettings) (Devereux et al.,(1984) Nucleic Acids Res. 12: 387-395). ThePILEUP and BLAST algorithms can be used to calculate homology or line upsequences (typically on their default settings), for example asdescribed in Altschul (1993) J. Mol. Evol. 36: 290-300; Altschul et al(1990) J. Mol. Biol. 215: 403-410.

[0059] Software for performing BLAST analyses is publicly availablethrough the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying highscoring sequence pair (HSPs) by identifying short words of length W inthe query sequence that either match or satisfy some positive-valuedthreshold score T when aligned with a word of the same length in adatabase sequence. T is referred to as the neighborhood word scorethreshold (Altschul et al. 1990). These initial neighborhood word hitsact as seeds for initiating searches to find HSPs containing them. Theword hits are extended in both directions along each sequence for as faras the cumulative alignment score can be increased. Extensions for theword hits in each direction are halted when: the cumulative alignmentscore falls off by the quantity X from its maximum achieved value; thecumulative score goes to zero or below, due to the accumulation of oneor more negative-scoring residue alignments; or the end of eithersequence is reached. The BLAST algorithm parameters W, T and X determinethe sensitivity and speed of the alignment. The BLAST program uses asdefaults a word length (W) of 11, the BLOSUM62 scoring matrix (seeHenikoff and Henikoff (1992) Proc. Natl. Acad. Sci. USA 89: 10915-10919)alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparisonof both strands.

[0060] The BLAST algorithm performs a statistical analysis of thesimilarity between two sequences; see e.g., Karlin and Altschul (1993)Proc. Natl. Acad. Sci. USA 90: 5873-5787. One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a sequence is considered similar to another sequence if thesmallest sum probability in comparison of the first sequence to thesecond sequence is less than about 1, preferably less than about 0.1,more preferably less than about 0.01, and most preferably less thanabout 0.001.

[0061] Any combination of the above mentioned degrees of sequenceidentity and minimum sizes may be used to define polynucleotides of theinvention, with the more stringent combinations (i.e. higher sequenceidentity over longer lengths) being preferred. Thus, for example apolynucleotide which has at least 90% sequence identity over 25,preferably over 30 nucleotides forms one aspect of the invention, asdoes a polynucleotide which has at least 95% sequence identity over 40nucleotides.

[0062] The nucleotides according to the invention have utility inproduction of the polypeptides according to the invention. Producitonmay take place in vitro, in vivo or ex vivo. The nucleotides may beinvolved in recombinant protein synthesis, as therapeutic agents intheir own right, utilized in gene therapy techniques, and/or utilized inthe production of nonhuman transgenic animals. Nucleotides complementaryto those encoding PXR, or antisense sequences, may also be usedtherapeutically.

[0063] Polynucleotides of the invention may be used as primers, e.g. PCRprimers or primers for an alternative amplification reaction, or asprobes, e.g. polynucleotides detectably labeled by conventional meansusing radioactive or non-radioactive labels. In addition, thepolynucleotides may be cloned into vectors.

[0064] Such primers, probes and other fragments will preferably be atleast 10, preferably at least 15 or at least 20, for example at least25, at least 30 or at least 40 nucleotides in length. They willtypically be up to 40, 50, 60, 70, 100 or 150 nucleotides in length.Probes and fragments can be longer than 150 nucleotides in length, forexample up to 200, 300, 400, 500, 600, 700 nucleotides in length, oreven up to a few nucleotides, such as five or ten nucleotides, short ofthe coding sequence of SEQ ID NO: 1, 3, 5 or 7.

[0065] The polynucleotides of the present invention are also useful inthe production of chimeric receptors or fusion proteins having a PXRcomponent which comprises at least a DNA binding domain or a ligandbinding domain of a canine, primate, porcine or fish PXR and a non-PXRderived sequence. Non-PXR derived sequences can be selected so as to besuitable for the purpose to be served by the chimeric receptor. Examplesof such sequences include, but are not limited to,glutathione-S-transferase, the DNA binding domain of yeast transcriptionfactor GAL4 and other DNA binding domains such as the DNA bindingdomains for estrogen or glucocorticoid receptors, and the viral VP16transcriptional activation domain. Chimeric receptors of the presentinvention may further comprise a detectable label such as a radioactiveor fluorescent label. The chimeric receptors may also be bound to asolid support such as glass or plastic particles or plates or a filter.

[0066] The present invention also includes expression vectors thatcomprise nucleotide sequences encoding the polypeptides or variantsthereof of the invention. Such expression vectors are routinelyconstructed in the art of molecular biology and may for example involvethe use of plasmid DNA and appropriate initiators, promoters, enhancersand other elements, such as for example polyadenylation signals whichmay be necessary, and which are positioned in the correct orientation,in order to allow for protein expression. Other suitable vectors wouldbe apparent to persons skilled in the art based upon teachings providedherein and what is known in the art. By way of further example in thisregard we refer to Sambrook et al. 1989.

[0067] Polynucleotides according to the invention may also be insertedinto the vectors described above in an antisense orientation in order toprovide for the production of antisense RNA. Antisense RNA or otherantisense polynucleotides may also be produced by synthetic means. Suchantisense polynucleotides may be used as test compounds in the assays ofthe invention or may be useful therapeutically.

[0068] Preferably, a polynucleotide of the invention used in a vector isoperably linked to a control sequence which is capable of providing forthe expression of the coding sequence by the host cell, i.e. the vectoris an expression vector. The term “operably linked” refers to ajuxtaposition wherein the components described are in a relationshippermitting them to function in their intended manner. For example, aregulatory sequence, such as a promoter, “operably linked” to a codingsequence is positioned in such a way that expression of the codingsequence is achieved under conditions compatible with the regulatorysequence.

[0069] The vectors may be for example, plasmid, virus or phage vectorsprovided with an origin of replication, optionally a promoter for theexpression of the polynucleotide and optionally a regulator of thepromoter. The vectors may contain one or more selectable marker genes,for example an ampicillin resistance gene in the case of a bacterialplasmid or a resistance gene for a fungal vector. Vectors may be used invitro, for example, for the production of DNA or RNA or used totransfect or transform a host cell, for example, a mammalian host cell.The vectors may also be adapted to be used in vivo, for example in amethod of gene therapy or in the production of nonhuman transgenicanimals, preferably mice. Additional vector components known in the artare suitable for use in the vectors of the present invention andinclude, for example, processing sites such as a polyadenylation signal,ribosome binding sites, RNA splice sites, and transcriptionaltermination sequences.

[0070] Promoters and other expression regulation signals may be selectedto be compatible with the host cell for which expression is designed.Examples of yeast promoters which can be used in the present inventioninclude S. cerevisiae GAL4 and ADH promoters, and S. pombe NMT1 and ADHpromoters. Viral promoters can also be used. Examples of viral promotersinclude, but are not limited to, the Moloney murine leukemia virus longterminal repeat (MMLV LTR), the Rous sarcoma virus (RSV) LTR promoter,the SV40 promoter, the human cytomegalovirus (CMV) IE promoter,adenovirus, HSV promoters (such as the HSV IE promoters), or HPVpromoters, particularly the HPV upstream regulatory region (URR). Amammalian promoter useful in the present invention is themetallothionein promoter which can be induced in response to heavymetals such as cadmium and β-actin promoters. Tissue-specific promotersare especially preferred. All these promoters, as well as additionalpromoters useful in the present invention, are readily available in theart.

[0071] The vector may further include sequences flanking thepolynucleotide giving rise to polynucleotides which comprise sequenceshomologous to eukaryotic genomic sequences, preferably mammalian genomicsequences or viral genomic sequences. This allows the introduction ofthe polynucleotides of the invention into the genome of eukaryotic cellsor viruses by homologous recombination. In particular, a plasmid vectorcomprising the expression cassette flanked by viral sequences can beused to prepare a viral vector suitable for delivering thepolynucleotides of the invention to a mammalian cell. Other examples ofsuitable viral vectors include herpes simplex viral vectors andretroviruses, including lentiviruses, adenoviruses, adeno-associatedviruses and HPV viruses. Gene transfer techniques using these virusesare known to those skilled in the art. Retrovirus vectors for examplemay be used to stably integrate the polynucleotide into the host genome.Replication-defective adenovirus vectors by contrast remain episomal andtherefore allow transient expression.

[0072] The invention also includes cells that have been modified toexpress the PXR polypeptide or a variant thereof. Such cells includetransient, or preferably stable higher eukaryotic cell lines such asmammalian cells or insect cells, lower eukaryotic cells such as yeast,or prokaryotic cells such as bacterial cells. Examples of cells whichmay be modified by insertion of vectors encoding for a polypeptideaccording to the invention include, but are not limited to, mammalianHEK293T, CHO, HeLa and COS cells.

[0073] A polypeptide of the invention may also be expressed in cells ofa transgenic non-human animal, preferably a mouse. Accordingly,transgenic non-human animals expressing a PXR polypeptide of theinvention are also included within the scope of the invention. Forexample, transgenic mice can be generated that express a selected PXR ofthe present invention as well as the endogenous mouse PXR gene. Mice canalso be generated in which the endogenous PXR gene is knocked out andthen replaced by the selected PXR polynucleotide of the presentinvention. Transgenic animals can also be generated that expressisoforms of a selected PXR as well as mutant alleles of the PXRs of thepresent invention. Transgenic animals developed by these methods can beused to screen compounds for drug interactions and toxicities and tostudy the regulation of P450 3A4 in vivo.

[0074] According to another aspect, the present invention also relatesto antibodies, specific for a polypeptide of the invention. Suchantibodies are for example useful in purification, isolation orscreening methods involving immunoprecipitation techniques or, indeed,as therapeutic agents in their own right.

[0075] Antibodies can be raised against specific epitopes of thepolypeptides according to the invention. Such antibodies may be used toblock ligand binding to the receptor. An antibody, or other compound,“specifically binds” to a protein when it binds with preferential orhigh affinity to the protein or polypeptide for which it is specific butdoes not substantially bind or binds with only low affinity to otherproteins. A variety of protocols for competitive binding orimmunoradiometric assays to determine the specific binding capability ofan antibody are well known in the art (see for example Maddox et al,(1993) J. Exp. Med. 158: 1211-1226). Such immunoassays typically involvethe formation of complexes between the specific protein and its antibodyand the measurement of complex formation.

[0076] Antibodies of the invention may be antibodies to the canine,primate, porcine or fish polypeptides or fragments thereof. For thepurposes of this invention, the term “antibody”, unless specified to thecontrary, includes fragments which bind a polypeptide of the invention.Such fragments include Fv, F(ab′) and F(ab′)₂ fragments, as well assingle chain antibodies. Furthermore, the antibodies and fragmentsthereof may be chimeric antibodies, CDR-grafted antibodies or humanizedantibodies.

[0077] Antibodies may be used in methods for detecting polypeptides ofthe invention in a biological sample. In these methods, an antibody ofthe invention is first provided. A biological sample is then incubatedwith the antibody under conditions which allow for the formation of acomplex between the antibody and the polypeptide or antigen and theamount of antibody-polypeptide complex formed is determined. Variousmethods for determining formation of an antibody-antigen complex arewell known to those of skill in the art.

[0078] For purposes of the present invention, by “biological sample” itis meant to include, but is not limited to, tissue extracts, blood,serum, saliva, urine, cerebral spinal fluid, and bile.

[0079] Antibodies of the invention may be bound to a solid supportand/or packaged into kits in a suitable container along with suitablereagents, controls, instructions, etc. Antibodies may be linked to arevealing label and thus may be suitable for use in methods of in vivoPXR imaging.

[0080] Antibodies of the invention can be produced by any suitablemethod. Means for preparing and characterizing antibodies are well knownin the art, see for example Harlow and Lane (1988) “Antibodies: ALaboratory Manual”, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. For example, an antibody may be produced by raisingantibody in a host animal against the whole polypeptide or a fragmentthereof, for example an antigenic epitope thereof, herein after the“immunogen”.

[0081] A method for producing a polyclonal antibody comprises immunizinga suitable host animal, for example an experimental animal, with theimmunogen and isolating immunoglobulins from the animal's serum. In thismethod, the animal is inoculated with the immunogen. Blood issubsequently removed from the animal and the IgG fraction purified.

[0082] A method for producing a monoclonal antibody comprisesimmortalizing cells which produce the desired antibody. Hybridoma cellscan be produced by fusing spleen cells from an inoculated experimentalanimal with tumor cells (Kohler and Milstein (1975) Nature 256:495-497). An immortalized cell producing the desired antibody may beselected by a conventional procedure. Hybridomas are then grown inculture or injected intraperitoneally for formation of ascites fluid orinto the blood stream of an allogenic host or immunocompromised host.

[0083] For the production of both monoclonal and polyclonal antibodies,the experimental animal is suitably a goat, rabbit, rat or mouse. Ifdesired, the immunogen may be administered as a conjugate in which theimmunogen is coupled, for example via a side chain of one of the aminoacid residues, to a suitable carrier. The carrier molecule is typicallya physiologically acceptable carrier. The antibody obtained may beisolated and, if desired, purified.

[0084] A further aspect of the present invention relates to in vitro(cell-free) and in vivo (cell-based) assays that can be used to profilethe effects of compounds, particularly potential new drugs, on P450 3A4levels in various species. These assays can take any of a variety offorms. Since compounds that activate PXR function are inducers of P4503A4 gene expression, binding and activation assays using selected PXRsof the present invention provide efficient means to identify compoundsexpected to activate P450 3A4 in a selected species.

[0085] Binding assays of the present invention include cell free assaysin which a selected PXR, or the ligand binding domain of a selected PXR(alone or present as a fusion protein), is incubated with a testcompound which, advantageously, bears a detectable label (e.g. aradioactive or fluorescent label). The selected PXR, or ligand bindingdomain thereof, free or bound to the test compound, is then separatedfrom free test compound using any of variety of techniques (e.g., usinggel filtration chromatography (for example, on Sephadex G50 spincolumns) or through capture on a hydroxyapatite resin). The amount oftest compound bound to the selected PXR or ligand binding domainthereof, is then determined via detection of the label.

[0086] An alternative approach for detecting radiolabeled test compoundbound to a selected PXR, or ligand binding domain thereof, is ascintillation proximity assay (SPA). In this assay, a bead (or otherparticle) is impregnated with scintillant and coated with a moleculethat can capture the selected PXR, or ligand binding domain thereof(e.g., streptavidin-coated beads can be used to capture biotinylated PXRligand binding domain). Radioactive counts are detected only when thecomplex of radiolabeled test compound and the selected PXR, or ligandbinding domain thereof, is captured on the surface of the SPA beadbringing the radioactive label into sufficient proximity to thescintillant to emit a signal.

[0087] This approach has the advantage of not requiring the separationof free test compound from bound (Nichols et al, Anal. Biochem.257:112-119 (1998)).

[0088] Assays to determine whether a test compound interacts with aselected PXR ligand binding domain can also be performed via acompetition binding assay. In this assay, the selected PXR, or ligandbinding domain thereof, is incubated with a compound known to interactwith the selected PXR, which compound, advantageously, bears adetectable label (e.g., a radioactive or fluorescent label). A testcompound is added to the reaction and assayed for its ability to competewith the labeled compound for binding to the selected PXR, or ligandbinding domain thereof. A standard assay format employing a step toseparate free known (labeled) compound from bound, or an SPA format, canbe used to assess the ability of the test compound to compete.

[0089] To determine if a test compound activates a selected PXR, andthus induces P450 3A4 expression, the ligand binding domain of theselected PXR is prepared (e.g., expressed) as a fusion protein (e.g.,with glutathione-S-transferase (GST), a histidine tag or a maltosebinding protein). The fusion protein and coactivator (either or bothadvantageously labeled with a detectable label, e.g., a radiolabel orfluorescent tag) are incubated in the presence and absence of the testcompound and the extent of binding of the coactivator to the fusionprotein determined. The induction of interaction in the presence of thetest compound is indicative of an activator of the selected PXR.

[0090] PXR activation assays in accordance with the invention can becarried out using a full length PXR and a reporter system comprising oneor more copies of the DNA binding site recognized by the PXR bindingdomain. More preferably, however, the activation assays are conductedusing established chimeric receptor systems. For example, the ligandbinding domain of a selected PXR can be fused to the DNA binding domainof, for example, yeast transcription factor GAL4, or that of theestrogen or glucocorticoid receptor. An expression vector for thechimera (e.g.,a GAL4-PXR chimera) can be transfected into host cells(e.g., CV-1, HuH7, HepG2 or Caco2 cells) together with a reportedconstruct. The reporter construct may comprise one or more (e.g., 5)copies of the DNA binding site recognized by the binding domain presentin the chimera (e.g., the GAL4 DNA binding site) driving expression of areporter gene (e.g., CAT, SPAP or luciferase). Cells containing theconstructs are then treated with either vehicle alone or vehiclecontaining test compound, and the level of expression of the reportergene determined. In accordance with this assay, enhancement ofexpression of the reporter gene in the presence of the test compoundindicates that the test compound activates the selected PXR and thus canfunction as an inducer of CYP3A4 gene expression in that species.

[0091] Another format suitable for use in connection with the presentinvention is the yeast two-hybrid assay. This is an established approachto detect protein-protein interactions that is performed in yeast.Protein #1, representing the bait, is expressed in yeast as a chimerawith a DNA binding domain (e.g., GAL4). Protein #2, representing thepredator, is expressed in the same yeast cell as a chimera with a strongtranscriptional activation domain. The interaction of bait and predatorresults in the activation of a reporter gene (e.g., luciferase orβ-galactosidase) or the regulation of a selectable marker (e.g., LEU2gene). This approach can be used as a screen to detect, for example,ligand-dependent interactions between a selected PXR and other proteinssuch as coactivator proteins (e.g., SRCI, TIFI, TIF2, ACTR) or fragmentsthereof (Fields et al., Nature 340:245-2.46 (1989)).

[0092] Still another format is the ligand-induced complex formation(LIC) assay. This assay detects ligand-mediated effects on nuclearreceptor-DNA interactions. A selected PXR (or, minimally, DNA and/orligand binding domains thereof) can be incubated with its heterodimericpartner RXR in the presence of DNA representing an established PXR/RXRbinding site. Test-compounds can be assayed for their ability to eitherenhance or interfere with binding of the PXR/RXR heterodimer to DNA(Forman et al, Proc. Natl. Acad. Sci. USA 94:4312-4317 (1997).

[0093] In a preferred embodiment, the screening assay takes the form ofa FRET (Fluorescence Resonance Emission Transfer assay (Nichols et al.(1998) Anal. Biochem. 257:112-119). This screening assay comprises thesteps of exposing a sample portion comprising the donor located at afirst position and the acceptor located at the second position to lightat a first wavelength capable of inducing a first electronic transitionin the donor. The donor comprises a complex of a lanthanide chelate anda lanthanide capable of binding the chelate. The spectral overlap of thedonor emission and acceptor absorption is sufficient to enable energytransfer from the donor to the acceptor as measured by a detectableincrease in acceptor luminescence. In a preferred embodiment, a SRC-1(LCD2, 677-696) lanthanide chelate is used. Preferably, the lanthanideelement comprises Europium and the signal chelate comprises Europiumbound to a PXR of the present invention. A signal pair comprisingEuropium bound to the PXR and APC (allophycocyanin) bound to SRC-1 (see,e.g., Parks et al. (1999) Science 284:1365-1368) can also be used.

[0094] Suitable test compounds which can be screened in the above assaysinclude combinatorial libraries, defined chemical entities andcompounds, peptide and peptide mimetics, oligonucleotides, naturalproduct libraries such as display libraries (e.g. phage displaylibraries), and antibody products.

[0095] Typically, organic molecules, preferably small organic moleculeswhich have a molecular weight of from 50 to 2500 daltons, are screened.Candidate test compounds can be biomolecules including, saccharides,fatty acids, steroids, purines, pyrimidines, derivatives, structuralanalogs and combinations thereof. Such test compounds are obtained froma wide variety of sources including libraries of synthetic and naturalcompounds. Further, known pharmacological agents can be subjected todirected or random chemical modifications, such as acylation,alkylation, esterification, amidification, etc. to produce structuralanalogs.

[0096] Test compounds can be used in an initial screen of, for example,10 compounds per reaction, and the compounds of these batches which showinhibition or activation re-screened individually. Test compounds may bescreened at a concentration of from 1 nM to 1000 μM, preferably from 1μM to 100 μM, more preferably from 1 μM to 10 μM. Preferably, theactivity of a test compound is compared to the activity shown by a knownactivator or inhibitor. A test compound which acts as an inhibitorpreferably produces a 50% inhibition of activity of the receptor.Alternatively a test compound which acts as an activator preferablyproduces 50% of the maximal activity produced using a known activator.

[0097] Comparative pharmacology involves the use of a nonhuman animalmodel to either predict the effects of a compound in humans, or providea contrast to the effects of a compound in humans. Accordingly, resultsfrom assays such as described above with the PXRs of the presentinvention provide important information with respect to whether acompound of interest modulates the receptor similarly to or differentlyfrom the analogous human receptor.

[0098] Further, comparison of activation of human PXRs with activationof a non-human PXR of the present invention or PXRs from other nonhumananimals which are used as models in preclinical studies is useful inselection of preclinical animal models predictive of affects of a testcompound on P450 3A4 in humans. In this method, in vitro activation ofhuman PXR in the presence of a test compound is compared with in vitroactivation of PXRs from various preclinical animal models, including,but not limited to the PXRs of the present invention, in the presence ofthe same test compound. A PXR from a preclinical animal model exhibitingsimilar in vitro activation to the human PXR in the presence of the testcompound is indicative of the preclinical animal model being predictiveof the affects of the test compound on P450 3A4 in humans.

[0099] Activation of the nonhuman PXRs of the present invention wasexamined in the presence of various steroids, xenobiotics, and bileacids. Steroids and xenobiotics screened for effects on activation ofthe nonhuman PXRs of the present invention included: pregnenolone16a-carbonitrile (PCN); rifampicine; 3 amino ethyl benzoate; TCPOBOP(1,4-bis[2-(3,5-dichloropyridyloxy)]benzene); epoxycholesterol; RU 486;omeprazole; primidone; ethosuximide; nifedipine; metyrapone; reserpine;trans-nanochlor; androstanol; clofibrate; clofibric acid; troglitazone;6, 16-dimethylpregnenolone; pregnenolone; 17a-OH pregnenolone;progesterone; 17a-OH-progesterone 5b-pregnane 3,20-dione;corticosterone; cortisone; DHEA (dehydroepiandrosterone); DHT(dihydrotestosterone); spironolactone; b-estradiol; tamoxifen;dexamethasone; dex-t-butylacetate; hydrocortisone; d-aldosterone;cyproterone acetate; hyperforin; phenobarbital; carbamazepine;phenytoin; clotrimazole; SR12813; lovastatin; mevastatin; squalestatin;and chlorpromazine. Bile acids screened for effects on activation of thenonhuman PXRs of the present invention included: 12-ketolithocholicacid; 3,6-diketocholanic acid; 3,7-diketocholanic acid; 3a,7a-dihydroxy-12-ketocholanic acid; 6-ketolithocholic acid;7,12-diketolithocholic acid; 7-ketodeoxycholic acid; 7-ketolithocholicacid; chenodeoxycholic acid; cholic acid; dehydrolithocholic acid;deoxycholic acid; 7-ketodeoxycholic acid methyl ester;glychochenodeoxycholic acid; glycocholic acid; glycodehydrocholic acid;glycodeoxycholic acid; glycohyocholic acid; glychohyodeoxycholic acid;taurodeoxycholic acid; glycolithocholic acid; hyocholic acid;hyodeoxycholic acid; lithocholic acid; murocholic acid;taurochenodeoxycholic acid; taurocholanic acid; taurocholic acid;taurodehydrocholic acid; taurohyocholic acid; taurodeoxycholic acid;taurolithocholic acid; tauro-b-muricholic acid; ursocholanic acid;ursodeoxycholic acid; a-muricholic acid; b-muricholic acid; 5b-cholanicacid-7a,12a-diol-3-one; and 5b-cholanic acid-3,7,12-trione. Theco-transactivation assay described in Example 2 was used to screen thesecompounds. Activation was assessed via measurement of levels of secretedplacental alkaline phosphates normalized (normalized SPAP) totransfection levels of a control gene. Results from these experimentsare depicted in FIGS. 1-8. Xenobiotics and steroids were assessed at aconcentration of 10 μM unless otherwise indicated on the graph. Bileacids were assessed at a concentration of 100 μM unless otherwiseindicated on the graph.

[0100] As can be seen from these experiments, PXRs from differentspecies exhibited varying activation patterns in the presence of thesame compounds. Similar activation screening assays to these can beperformed with other test compounds, most preferably new drugs indevelopment. Results from these assays are useful in PXR comparativepharmacology and selecting appropriate animal models for preclinicalstudies predictive of effects in humans.

[0101] The following nonlimiting examples further illustrate the presentinvention.

EXAMPLES Example 1 Characterization of the Sequence

[0102] Four PXR LBD sequences from pig, dog, zebrafish, and rhesusmonkey were cloned. The isolation of each sequence was achieved usingessentially the same strategy for each. A small stretch of the LBD wasobtained using either cross-hybridizing PCR primers from anotherspecies, or by finding some portion of the LBD sequence in the ESTdatabase. The remainder of the LBD was subsequently isolated by PCRamplification of flanking sequence using a primer from within thestarting sequence combined with either A) a degenerate oligorepresenting the canonical P-box of the DBD to isolate 5′ sequence, orB) oligo d(T)₂₀-G, d(T)₂₀-C,or d(T)₂₀-A to isolate 3′ sequence. Afterderiving the sequence to the poly (A) tail, the full-length LBD wasproduced using primers flanking the coding sequence. Wild-type sequencewas determined through examination of at least three independentamplifications of each full-length LBD.

[0103] To clone pig PXR LBD, total mRNA was prepared from frozen pigliver (1g) using the FastTrack 2.0 RNA Preparation kit (InVitrogen, SanDiego, Calif.). Oligo d(T)-primed cDNA synthesis was carried out byRT-PCR using a cDNA Cycle Kit (InVitrogen, San Diego, Calif.). Anapproximately 250 base pair stretch of pig PXR LBD was amplified fromthis cDNA using homologous mouse PXR LBD primers.

[0104] To clone dog PXR LBD, human PXR LBD primers were used to amplifyan approximately 450 base pair fragment from a dog liver 5′-stretchλgt11 cDNA library (Clontech, Palo Alto, Calif.).

[0105] To clone rhesus PXR LBD, human primers were used to amplify allbut the termini of the rhesus PXR LBD from a rhesus liver cDNA library.Primer sequences 5′-TGC CGT GTA TGT GGG GAC AAG GC-3′ (SEQ ID NO:9) and5′-GGC ATG AAG AA GAG ATG ATC ATG-3′ (SEQ ID NO:10) were used to amplifya 274 base pair fragment from a Rhesus liver library constructed in theCMVSport6 vector. The fragment was sequenced and new primers weredesigned based on this Rhesus sequence. These primers were then usedwith vector arm primers to amplify the regions 5′ and 3′ of the knownsequenced fragment.

[0106] To clone zebrafish (Danio rerio) PXR LBD, an initial fragment ofthe PXR LBD was identified as an EST sequence (Accession # AI943313).This sequence was used to design primers for amplification of the entireLBD from cDNA synthesized using zebrafish embryo (48h) oligo d(T)-primedcDNA.

Example 2 Characterization of Selected PXRs Via Cotransfection Assays

[0107] In order to assess ligands for their ability to activate PXR in acell-based assay, a transient transfection approach was utilized. PXRligand binding domains were fused to the Gal4 DNA binding domain andtested against a reporter gene regulated by a Gal4 response element(from the yeast UAS_(G)). Lipofectamine-based transient transfectionassays were conducted as described previously (Jones et al., 2000Molecular Endocrinology, volume 14, pp. 27-39), except that aUAS-tk-SPAP reporter vector was used instead of the (CYP3A1 DR3)₂-tk-CATreporter vector. When testing bile acids, an expression plasmid encodingintestinal bile acid transporter (IBAT) was added to facilitate cellularuptake of bile acids.

1 8 1 990 DNA Canine CDS (1)...(990) 1 ggc atg aag aag gag atg atc atgtcc gac gcg gct gtg gag cag agg 48 Gly Met Lys Lys Glu Met Ile Met SerAsp Ala Ala Val Glu Gln Arg 1 5 10 15 cgg gct ctg atc cgg agg aaa aagcga gaa cgg atg ggc gcg tcg ccg 96 Arg Ala Leu Ile Arg Arg Lys Lys ArgGlu Arg Met Gly Ala Ser Pro 20 25 30 ctg gga gcc aag ggg ctg agt gag gagcag cag acg atg atc cga gag 144 Leu Gly Ala Lys Gly Leu Ser Glu Glu GlnGln Thr Met Ile Arg Glu 35 40 45 ctg atg gat gcc cag atg aaa acc ttt gacacc acc ttc tcc aac ttc 192 Leu Met Asp Ala Gln Met Lys Thr Phe Asp ThrThr Phe Ser Asn Phe 50 55 60 aag gat ttc cgg ctg ccg gcc gcg tgc agc agcggg cgc gag gtc cca 240 Lys Asp Phe Arg Leu Pro Ala Ala Cys Ser Ser GlyArg Glu Val Pro 65 70 75 80 gga gcg gcg cac act cca gtg ggg gag gaa gctgcc aag tgg agc cag 288 Gly Ala Ala His Thr Pro Val Gly Glu Glu Ala AlaLys Trp Ser Gln 85 90 95 gtc agg gag gat ctg tgc tcg ctg aag gtg tgc ctgcgg ctg cgc ggg 336 Val Arg Glu Asp Leu Cys Ser Leu Lys Val Cys Leu ArgLeu Arg Gly 100 105 110 gag gac ggc agc gtc cag aac tac aca ccc cag gccgac cgc agc ggc 384 Glu Asp Gly Ser Val Gln Asn Tyr Thr Pro Gln Ala AspArg Ser Gly 115 120 125 gcc gag atc ttt tcc ctg ctg ccc cac atg gct gacatg tcc acc tac 432 Ala Glu Ile Phe Ser Leu Leu Pro His Met Ala Asp MetSer Thr Tyr 130 135 140 atg ttc aaa ggc gtc atc aac ttt gcc aaa gtc atctcc cac ttc agg 480 Met Phe Lys Gly Val Ile Asn Phe Ala Lys Val Ile SerHis Phe Arg 145 150 155 160 gaa ttg ccc atc gag gac cag atc tcg ctg ctaaag ggg gcc acc ttc 528 Glu Leu Pro Ile Glu Asp Gln Ile Ser Leu Leu LysGly Ala Thr Phe 165 170 175 gag gtg tgc cag ctg agg ttc aac acg gtg ttcaac gca gag acc gga 576 Glu Val Cys Gln Leu Arg Phe Asn Thr Val Phe AsnAla Glu Thr Gly 180 185 190 acc tgg gag tgt ggc cgg ctg tcc tac tgc ctggaa gac cct gca ggc 624 Thr Trp Glu Cys Gly Arg Leu Ser Tyr Cys Leu GluAsp Pro Ala Gly 195 200 205 ggc ttc cag cag ctt ctc ctg gag cca gtg ctgaag ttc cac tac agg 672 Gly Phe Gln Gln Leu Leu Leu Glu Pro Val Leu LysPhe His Tyr Arg 210 215 220 ctg aag agg ctg cag ctg cat aag gag gag tatgtg ctg atg cag gcc 720 Leu Lys Arg Leu Gln Leu His Lys Glu Glu Tyr ValLeu Met Gln Ala 225 230 235 240 atc tct ctt ttc tcc cca gac cgc cca ggtgtg gtg cag cgc agc gtg 768 Ile Ser Leu Phe Ser Pro Asp Arg Pro Gly ValVal Gln Arg Ser Val 245 250 255 gtg gac cag ctg cag gag aga ttt gcc atcgcc ctg aag gcc tac atc 816 Val Asp Gln Leu Gln Glu Arg Phe Ala Ile AlaLeu Lys Ala Tyr Ile 260 265 270 gag tgc aat cgg ccg cag cct gcc cac cggttc ctg ttc ctg aag atc 864 Glu Cys Asn Arg Pro Gln Pro Ala His Arg PheLeu Phe Leu Lys Ile 275 280 285 atg gcc atg ctc acc gag ctt cgc agc atcaat gcc cag cac acc cag 912 Met Ala Met Leu Thr Glu Leu Arg Ser Ile AsnAla Gln His Thr Gln 290 295 300 aag ctg ctg cgc atc cag gac ata cac cccttc gcc agc ccc ctc atg 960 Lys Leu Leu Arg Ile Gln Asp Ile His Pro PheAla Ser Pro Leu Met 305 310 315 320 cag gag ctg ttc agc atc acg gac ggctga 990 Gln Glu Leu Phe Ser Ile Thr Asp Gly * 325 2 329 PRT Canine 2 GlyMet Lys Lys Glu Met Ile Met Ser Asp Ala Ala Val Glu Gln Arg 1 5 10 15Arg Ala Leu Ile Arg Arg Lys Lys Arg Glu Arg Met Gly Ala Ser Pro 20 25 30Leu Gly Ala Lys Gly Leu Ser Glu Glu Gln Gln Thr Met Ile Arg Glu 35 40 45Leu Met Asp Ala Gln Met Lys Thr Phe Asp Thr Thr Phe Ser Asn Phe 50 55 60Lys Asp Phe Arg Leu Pro Ala Ala Cys Ser Ser Gly Arg Glu Val Pro 65 70 7580 Gly Ala Ala His Thr Pro Val Gly Glu Glu Ala Ala Lys Trp Ser Gln 85 9095 Val Arg Glu Asp Leu Cys Ser Leu Lys Val Cys Leu Arg Leu Arg Gly 100105 110 Glu Asp Gly Ser Val Gln Asn Tyr Thr Pro Gln Ala Asp Arg Ser Gly115 120 125 Ala Glu Ile Phe Ser Leu Leu Pro His Met Ala Asp Met Ser ThrTyr 130 135 140 Met Phe Lys Gly Val Ile Asn Phe Ala Lys Val Ile Ser HisPhe Arg 145 150 155 160 Glu Leu Pro Ile Glu Asp Gln Ile Ser Leu Leu LysGly Ala Thr Phe 165 170 175 Glu Val Cys Gln Leu Arg Phe Asn Thr Val PheAsn Ala Glu Thr Gly 180 185 190 Thr Trp Glu Cys Gly Arg Leu Ser Tyr CysLeu Glu Asp Pro Ala Gly 195 200 205 Gly Phe Gln Gln Leu Leu Leu Glu ProVal Leu Lys Phe His Tyr Arg 210 215 220 Leu Lys Arg Leu Gln Leu His LysGlu Glu Tyr Val Leu Met Gln Ala 225 230 235 240 Ile Ser Leu Phe Ser ProAsp Arg Pro Gly Val Val Gln Arg Ser Val 245 250 255 Val Asp Gln Leu GlnGlu Arg Phe Ala Ile Ala Leu Lys Ala Tyr Ile 260 265 270 Glu Cys Asn ArgPro Gln Pro Ala His Arg Phe Leu Phe Leu Lys Ile 275 280 285 Met Ala MetLeu Thr Glu Leu Arg Ser Ile Asn Ala Gln His Thr Gln 290 295 300 Lys LeuLeu Arg Ile Gln Asp Ile His Pro Phe Ala Ser Pro Leu Met 305 310 315 320Gln Glu Leu Phe Ser Ile Thr Asp Gly 325 3 1441 DNA Primate PXR CDS(119)...(1423) 3 tccttggtaa agctactcct tgatcgatcc tttgcacctg attgttcaaagtggacccca 60 gggggaagtc agagcgaaga acttaccgct aagcagtcca agaggcccagaagcaaac 118 ctg gag gtg aga ccc aaa gaa ggc tgg aac cat gct gac ttt gtatac 166 Leu Glu Val Arg Pro Lys Glu Gly Trp Asn His Ala Asp Phe Val Tyr1 5 10 15 tgt gag gac aca gag ttt gct cct gga aag ccc act gtc aac gcagat 214 Cys Glu Asp Thr Glu Phe Ala Pro Gly Lys Pro Thr Val Asn Ala Asp20 25 30 gag gaa gtt ggg ggt ccc caa atc tgc cgt gta tgt ggg gac aag gcc262 Glu Glu Val Gly Gly Pro Gln Ile Cys Arg Val Cys Gly Asp Lys Ala 3540 45 act ggt tat cac ttc aat gtc atg aca tgt gaa gga tgc aag ggc ttt310 Thr Gly Tyr His Phe Asn Val Met Thr Cys Glu Gly Cys Lys Gly Phe 5055 60 ttc agg agg gcc atg aaa cgc aac gcc cgc ctt agg tgc ccc ttc cgg358 Phe Arg Arg Ala Met Lys Arg Asn Ala Arg Leu Arg Cys Pro Phe Arg 6570 75 80 aag ggc gcc tgc gag atc acc cgg aag acc cgg cga cag tgc cag gcc406 Lys Gly Ala Cys Glu Ile Thr Arg Lys Thr Arg Arg Gln Cys Gln Ala 8590 95 tgc cgg ctg cgc aag tgc ctg gag agc ggc atg aag aag gag atg atc454 Cys Arg Leu Arg Lys Cys Leu Glu Ser Gly Met Lys Lys Glu Met Ile 100105 110 atg tcc gac gcg gcc gta gag gag agg cgg gcc ttg atc aag agg aag502 Met Ser Asp Ala Ala Val Glu Glu Arg Arg Ala Leu Ile Lys Arg Lys 115120 125 aaa aga gaa cgg atc ggg act cag cca ccc gga gtg cag ggg ctg acg550 Lys Arg Glu Arg Ile Gly Thr Gln Pro Pro Gly Val Gln Gly Leu Thr 130135 140 gag gag cag cgg atg atg atc agg gag ctg atg gac gct cag atg aaa598 Glu Glu Gln Arg Met Met Ile Arg Glu Leu Met Asp Ala Gln Met Lys 145150 155 160 acc ttt gac act acc ttc tcc cat ttc aag aat ttc cgg ctg ccaggg 646 Thr Phe Asp Thr Thr Phe Ser His Phe Lys Asn Phe Arg Leu Pro Gly165 170 175 gtg ctt agc agt ggc tgt gag atg cca gag tct ctg cag gcc ccatcg 694 Val Leu Ser Ser Gly Cys Glu Met Pro Glu Ser Leu Gln Ala Pro Ser180 185 190 agg gaa gaa gct gcc aag tgg aac cag gtc agg aaa gat ctg tggtct 742 Arg Glu Glu Ala Ala Lys Trp Asn Gln Val Arg Lys Asp Leu Trp Ser195 200 205 gtg aag gtc tcc gtg cag ctg cgg ggg gag gat ggc agt gtc tggaac 790 Val Lys Val Ser Val Gln Leu Arg Gly Glu Asp Gly Ser Val Trp Asn210 215 220 tac aaa ccc cca gcc gac aat ggc ggg aaa gag atc ttc tcc ttgctg 838 Tyr Lys Pro Pro Ala Asp Asn Gly Gly Lys Glu Ile Phe Ser Leu Leu225 230 235 240 ccc cac atg gct gac atg tca acc tac atg ttc aaa ggc atcatc aac 886 Pro His Met Ala Asp Met Ser Thr Tyr Met Phe Lys Gly Ile IleAsn 245 250 255 ttt gcc aaa gtc atc tcc tac ttc agg gac ctg ccc atc gaggac cag 934 Phe Ala Lys Val Ile Ser Tyr Phe Arg Asp Leu Pro Ile Glu AspGln 260 265 270 atc tcc cta ctg aag ggg gcc act ttt gag ctg tgc cag ctgaga ttc 982 Ile Ser Leu Leu Lys Gly Ala Thr Phe Glu Leu Cys Gln Leu ArgPhe 275 280 285 aac aca gta ttc aac gcg gag act gga act tgg gag tgt ggccgg ctg 1030 Asn Thr Val Phe Asn Ala Glu Thr Gly Thr Trp Glu Cys Gly ArgLeu 290 295 300 tcc tac tgc ttg gaa gac cct gca ggt ggt ttc cag caa cttctg ctg 1078 Ser Tyr Cys Leu Glu Asp Pro Ala Gly Gly Phe Gln Gln Leu LeuLeu 305 310 315 320 gag ccc atg ctg aaa ttc cac tac atg ctg aag aag ctgcag cta cac 1126 Glu Pro Met Leu Lys Phe His Tyr Met Leu Lys Lys Leu GlnLeu His 325 330 335 gag gag gag tat gtg ctg atg cag gcc atc tcc ctc ttctcc cca gac 1174 Glu Glu Glu Tyr Val Leu Met Gln Ala Ile Ser Leu Phe SerPro Asp 340 345 350 cgc cca ggt gtg gtg cag cac cgc gtg gtg gac cag ctgcag gag caa 1222 Arg Pro Gly Val Val Gln His Arg Val Val Asp Gln Leu GlnGlu Gln 355 360 365 tac gct att act ctg aag tcc tac att gaa tgc aat cggccc cag cct 1270 Tyr Ala Ile Thr Leu Lys Ser Tyr Ile Glu Cys Asn Arg ProGln Pro 370 375 380 gct cat agg ttc ctg ttc ctg aag atc atg gct atg ctcacc gag ctc 1318 Ala His Arg Phe Leu Phe Leu Lys Ile Met Ala Met Leu ThrGlu Leu 385 390 395 400 cgc agc atc aac gcc cag cac acc cag cgg ctg ctgcgc atc cag gac 1366 Arg Ser Ile Asn Ala Gln His Thr Gln Arg Leu Leu ArgIle Gln Asp 405 410 415 ata cac ccc ttt gct acg ccc ctc atg cag gag ttgttc ggc atc acg 1414 Ile His Pro Phe Ala Thr Pro Leu Met Gln Glu Leu PheGly Ile Thr 420 425 430 ggt agc tga gtggctgccc ttgggtga 1441 Gly Ser * 4434 PRT Primate PXR 4 Leu Glu Val Arg Pro Lys Glu Gly Trp Asn His AlaAsp Phe Val Tyr 1 5 10 15 Cys Glu Asp Thr Glu Phe Ala Pro Gly Lys ProThr Val Asn Ala Asp 20 25 30 Glu Glu Val Gly Gly Pro Gln Ile Cys Arg ValCys Gly Asp Lys Ala 35 40 45 Thr Gly Tyr His Phe Asn Val Met Thr Cys GluGly Cys Lys Gly Phe 50 55 60 Phe Arg Arg Ala Met Lys Arg Asn Ala Arg LeuArg Cys Pro Phe Arg 65 70 75 80 Lys Gly Ala Cys Glu Ile Thr Arg Lys ThrArg Arg Gln Cys Gln Ala 85 90 95 Cys Arg Leu Arg Lys Cys Leu Glu Ser GlyMet Lys Lys Glu Met Ile 100 105 110 Met Ser Asp Ala Ala Val Glu Glu ArgArg Ala Leu Ile Lys Arg Lys 115 120 125 Lys Arg Glu Arg Ile Gly Thr GlnPro Pro Gly Val Gln Gly Leu Thr 130 135 140 Glu Glu Gln Arg Met Met IleArg Glu Leu Met Asp Ala Gln Met Lys 145 150 155 160 Thr Phe Asp Thr ThrPhe Ser His Phe Lys Asn Phe Arg Leu Pro Gly 165 170 175 Val Leu Ser SerGly Cys Glu Met Pro Glu Ser Leu Gln Ala Pro Ser 180 185 190 Arg Glu GluAla Ala Lys Trp Asn Gln Val Arg Lys Asp Leu Trp Ser 195 200 205 Val LysVal Ser Val Gln Leu Arg Gly Glu Asp Gly Ser Val Trp Asn 210 215 220 TyrLys Pro Pro Ala Asp Asn Gly Gly Lys Glu Ile Phe Ser Leu Leu 225 230 235240 Pro His Met Ala Asp Met Ser Thr Tyr Met Phe Lys Gly Ile Ile Asn 245250 255 Phe Ala Lys Val Ile Ser Tyr Phe Arg Asp Leu Pro Ile Glu Asp Gln260 265 270 Ile Ser Leu Leu Lys Gly Ala Thr Phe Glu Leu Cys Gln Leu ArgPhe 275 280 285 Asn Thr Val Phe Asn Ala Glu Thr Gly Thr Trp Glu Cys GlyArg Leu 290 295 300 Ser Tyr Cys Leu Glu Asp Pro Ala Gly Gly Phe Gln GlnLeu Leu Leu 305 310 315 320 Glu Pro Met Leu Lys Phe His Tyr Met Leu LysLys Leu Gln Leu His 325 330 335 Glu Glu Glu Tyr Val Leu Met Gln Ala IleSer Leu Phe Ser Pro Asp 340 345 350 Arg Pro Gly Val Val Gln His Arg ValVal Asp Gln Leu Gln Glu Gln 355 360 365 Tyr Ala Ile Thr Leu Lys Ser TyrIle Glu Cys Asn Arg Pro Gln Pro 370 375 380 Ala His Arg Phe Leu Phe LeuLys Ile Met Ala Met Leu Thr Glu Leu 385 390 395 400 Arg Ser Ile Asn AlaGln His Thr Gln Arg Leu Leu Arg Ile Gln Asp 405 410 415 Ile His Pro PheAla Thr Pro Leu Met Gln Glu Leu Phe Gly Ile Thr 420 425 430 Gly Ser 5993 DNA Porcine PXR CDS (1)...(993) 5 ggc atg agg aag gaa atg atc atgtca gat gca gct gtg gag cag agg 48 Gly Met Arg Lys Glu Met Ile Met SerAsp Ala Ala Val Glu Gln Arg 1 5 10 15 cgg gcc ttg atc agg agg aag aaacga gaa cag atc ggg gct cag ccc 96 Arg Ala Leu Ile Arg Arg Lys Lys ArgGlu Gln Ile Gly Ala Gln Pro 20 25 30 cca gga gcc aag ggt ctc act gaa gagcag cgg aca atg atc agt gag 144 Pro Gly Ala Lys Gly Leu Thr Glu Glu GlnArg Thr Met Ile Ser Glu 35 40 45 ctg atg aac gct cag atg aaa acc ttt gacacc acc ttc aca cat ttc 192 Leu Met Asn Ala Gln Met Lys Thr Phe Asp ThrThr Phe Thr His Phe 50 55 60 aag aat ttt cgg tta cca gag gtg ctt agc agtagc ctc gag att cca 240 Lys Asn Phe Arg Leu Pro Glu Val Leu Ser Ser SerLeu Glu Ile Pro 65 70 75 80 gag tgt ctg cag act ccg tcg tca agg gaa gaagct gcc aag tgg agc 288 Glu Cys Leu Gln Thr Pro Ser Ser Arg Glu Glu AlaAla Lys Trp Ser 85 90 95 aag ctc agg gaa gat ctg tgc tca gtg aaa ctc tctctg cag cta agg 336 Lys Leu Arg Glu Asp Leu Cys Ser Val Lys Leu Ser LeuGln Leu Arg 100 105 110 ggg gaa gat ggt agc gtc tgg aac tac aaa ccc ccagca gac aac agt 384 Gly Glu Asp Gly Ser Val Trp Asn Tyr Lys Pro Pro AlaAsp Asn Ser 115 120 125 ggg aaa gag atc ttt tcc ctg ctg ccc cac ata gctgac atg tca acc 432 Gly Lys Glu Ile Phe Ser Leu Leu Pro His Ile Ala AspMet Ser Thr 130 135 140 tac atg ttc aaa ggc att atc aac ttt gcc aaa gtcatc tcc tac ttc 480 Tyr Met Phe Lys Gly Ile Ile Asn Phe Ala Lys Val IleSer Tyr Phe 145 150 155 160 agg gac ttg ccc att gag gac cag atc tct ctgctg aag ggg gcc acc 528 Arg Asp Leu Pro Ile Glu Asp Gln Ile Ser Leu LeuLys Gly Ala Thr 165 170 175 ttt gag ctg tgc cag ctg aga ttc aac acg gtgttc aac gca gag acg 576 Phe Glu Leu Cys Gln Leu Arg Phe Asn Thr Val PheAsn Ala Glu Thr 180 185 190 ggg acc tgg gag tgt ggt cgg ctg tcc tac agcttg gaa gac ccc tca 624 Gly Thr Trp Glu Cys Gly Arg Leu Ser Tyr Ser LeuGlu Asp Pro Ser 195 200 205 ggt ggc ttc cag cag ctt ctc ctg cag ccc atgctg aaa ttc cac tac 672 Gly Gly Phe Gln Gln Leu Leu Leu Gln Pro Met LeuLys Phe His Tyr 210 215 220 atg ctg aag aag ctg cag ctg cat aag gag gagtat gtg ctg atg cag 720 Met Leu Lys Lys Leu Gln Leu His Lys Glu Glu TyrVal Leu Met Gln 225 230 235 240 gcc atc tcc ctt ttc tct cca gac cgc ccgggt gtg gtg caa cgc caa 768 Ala Ile Ser Leu Phe Ser Pro Asp Arg Pro GlyVal Val Gln Arg Gln 245 250 255 gtg gtg gac cag ctg cag gag agg ttt gccatt acc ctg aag gcc tac 816 Val Val Asp Gln Leu Gln Glu Arg Phe Ala IleThr Leu Lys Ala Tyr 260 265 270 atc gag tgc aac cgg ccc cag cct gcc caccga ttc ctg ttc ctg aag 864 Ile Glu Cys Asn Arg Pro Gln Pro Ala His ArgPhe Leu Phe Leu Lys 275 280 285 atc atg gct atg ctc act gag ctc cgc agcatc aac gcc caa cac acc 912 Ile Met Ala Met Leu Thr Glu Leu Arg Ser IleAsn Ala Gln His Thr 290 295 300 cag cgg ctg ctg cga atc cag gac ata cacccc ttc gcc acc cca ctc 960 Gln Arg Leu Leu Arg Ile Gln Asp Ile His ProPhe Ala Thr Pro Leu 305 310 315 320 atg cag gag tta ttc agc atc aca gaaagc tga 993 Met Gln Glu Leu Phe Ser Ile Thr Glu Ser * 325 330 6 330 PRTPorcine 6 Gly Met Arg Lys Glu Met Ile Met Ser Asp Ala Ala Val Glu GlnArg 1 5 10 15 Arg Ala Leu Ile Arg Arg Lys Lys Arg Glu Gln Ile Gly AlaGln Pro 20 25 30 Pro Gly Ala Lys Gly Leu Thr Glu Glu Gln Arg Thr Met IleSer Glu 35 40 45 Leu Met Asn Ala Gln Met Lys Thr Phe Asp Thr Thr Phe ThrHis Phe 50 55 60 Lys Asn Phe Arg Leu Pro Glu Val Leu Ser Ser Ser Leu GluIle Pro 65 70 75 80 Glu Cys Leu Gln Thr Pro Ser Ser Arg Glu Glu Ala AlaLys Trp Ser 85 90 95 Lys Leu Arg Glu Asp Leu Cys Ser Val Lys Leu Ser LeuGln Leu Arg 100 105 110 Gly Glu Asp Gly Ser Val Trp Asn Tyr Lys Pro ProAla Asp Asn Ser 115 120 125 Gly Lys Glu Ile Phe Ser Leu Leu Pro His IleAla Asp Met Ser Thr 130 135 140 Tyr Met Phe Lys Gly Ile Ile Asn Phe AlaLys Val Ile Ser Tyr Phe 145 150 155 160 Arg Asp Leu Pro Ile Glu Asp GlnIle Ser Leu Leu Lys Gly Ala Thr 165 170 175 Phe Glu Leu Cys Gln Leu ArgPhe Asn Thr Val Phe Asn Ala Glu Thr 180 185 190 Gly Thr Trp Glu Cys GlyArg Leu Ser Tyr Ser Leu Glu Asp Pro Ser 195 200 205 Gly Gly Phe Gln GlnLeu Leu Leu Gln Pro Met Leu Lys Phe His Tyr 210 215 220 Met Leu Lys LysLeu Gln Leu His Lys Glu Glu Tyr Val Leu Met Gln 225 230 235 240 Ala IleSer Leu Phe Ser Pro Asp Arg Pro Gly Val Val Gln Arg Gln 245 250 255 ValVal Asp Gln Leu Gln Glu Arg Phe Ala Ile Thr Leu Lys Ala Tyr 260 265 270Ile Glu Cys Asn Arg Pro Gln Pro Ala His Arg Phe Leu Phe Leu Lys 275 280285 Ile Met Ala Met Leu Thr Glu Leu Arg Ser Ile Asn Ala Gln His Thr 290295 300 Gln Arg Leu Leu Arg Ile Gln Asp Ile His Pro Phe Ala Thr Pro Leu305 310 315 320 Met Gln Glu Leu Phe Ser Ile Thr Glu Ser 325 330 7 1008DNA Zebrafish CDS (1)...(969) 7 ggc atg aag aga gag ctg atc atg tcg gatgag gcg gtg gag aag cgg 48 Gly Met Lys Arg Glu Leu Ile Met Ser Asp GluAla Val Glu Lys Arg 1 5 10 15 agg ttg cag atc agg agg aag agg atg caggaa gag cct gta act ctc 96 Arg Leu Gln Ile Arg Arg Lys Arg Met Gln GluGlu Pro Val Thr Leu 20 25 30 act cct caa cag gaa gct gtc ata caa gag ctgctg aac gca cac aag 144 Thr Pro Gln Gln Glu Ala Val Ile Gln Glu Leu LeuAsn Ala His Lys 35 40 45 aaa acc ttc gac atg act tgt gcc cat ttc agt cagttc cgg cct tta 192 Lys Thr Phe Asp Met Thr Cys Ala His Phe Ser Gln PheArg Pro Leu 50 55 60 gat cgg gat cag aag tct gtg tcc gag tcg agt cca ctcaca aac ggc 240 Asp Arg Asp Gln Lys Ser Val Ser Glu Ser Ser Pro Leu ThrAsn Gly 65 70 75 80 agc tgg atc gat cac aga ccc atc gct gaa gac cca atgcag tgg gtc 288 Ser Trp Ile Asp His Arg Pro Ile Ala Glu Asp Pro Met GlnTrp Val 85 90 95 ttc aat ccc act tcg ctc tcg tcc tct tcc tcc agc tac cagagc ctt 336 Phe Asn Pro Thr Ser Leu Ser Ser Ser Ser Ser Ser Tyr Gln SerLeu 100 105 110 gac aat aaa gag aag aag cac ttt aaa agt ggc aac ttc tcctct ctg 384 Asp Asn Lys Glu Lys Lys His Phe Lys Ser Gly Asn Phe Ser SerLeu 115 120 125 cca cac ttc aca gac ctc acc acg tac atg atc aag aat gtcatc aac 432 Pro His Phe Thr Asp Leu Thr Thr Tyr Met Ile Lys Asn Val IleAsn 130 135 140 ttc ggg aag acg ctg aca atg ttt agg gct ctg gtt atg gaggac cag 480 Phe Gly Lys Thr Leu Thr Met Phe Arg Ala Leu Val Met Glu AspGln 145 150 155 160 atc tcg ctg ctg aaa ggt gcc acc ttt gag atc att ctgatt cac ttc 528 Ile Ser Leu Leu Lys Gly Ala Thr Phe Glu Ile Ile Leu IleHis Phe 165 170 175 aac atg ttc ttt aat gaa gtg acg gga att tgg gag tgcggc ccc ctg 576 Asn Met Phe Phe Asn Glu Val Thr Gly Ile Trp Glu Cys GlyPro Leu 180 185 190 cag tac tgc atg gat gat gcc ttt cga gct ggt ttt cagcac cat ctg 624 Gln Tyr Cys Met Asp Asp Ala Phe Arg Ala Gly Phe Gln HisHis Leu 195 200 205 ctg gac cca atg atg aat ttc cat tac aca ctg cgt aagctg cgt ttg 672 Leu Asp Pro Met Met Asn Phe His Tyr Thr Leu Arg Lys LeuArg Leu 210 215 220 cat gag gag gag tat gtg ctg atg cag gcc ctc tct ctcttt tca cca 720 His Glu Glu Glu Tyr Val Leu Met Gln Ala Leu Ser Leu PheSer Pro 225 230 235 240 gat cgc cct ggt gtg aca gac cac aaa gtg atc gaccgc aac cag gaa 768 Asp Arg Pro Gly Val Thr Asp His Lys Val Ile Asp ArgAsn Gln Glu 245 250 255 aca cta gcg ctt acc cta aag act tac att gag gccaag aga aat ggg 816 Thr Leu Ala Leu Thr Leu Lys Thr Tyr Ile Glu Ala LysArg Asn Gly 260 265 270 cca gaa aaa cat ctg ctg ttc cca aag att atg gggtgc ctg acc gag 864 Pro Glu Lys His Leu Leu Phe Pro Lys Ile Met Gly CysLeu Thr Glu 275 280 285 atg agg agc atg aac gaa gag tac acc aaa caa gtgctg aaa atc cag 912 Met Arg Ser Met Asn Glu Glu Tyr Thr Lys Gln Val LeuLys Ile Gln 290 295 300 gac atg cag cct gaa gtg tct cca ctt tgg ttg gaaata ata agc aaa 960 Asp Met Gln Pro Glu Val Ser Pro Leu Trp Leu Glu IleIle Ser Lys 305 310 315 320 gac acc taa gtcctcacaa agcacgtgca actctacttattcccaact 1008 Asp Thr * 8 322 PRT Zebrafish 8 Gly Met Lys Arg Glu LeuIle Met Ser Asp Glu Ala Val Glu Lys Arg 1 5 10 15 Arg Leu Gln Ile ArgArg Lys Arg Met Gln Glu Glu Pro Val Thr Leu 20 25 30 Thr Pro Gln Gln GluAla Val Ile Gln Glu Leu Leu Asn Ala His Lys 35 40 45 Lys Thr Phe Asp MetThr Cys Ala His Phe Ser Gln Phe Arg Pro Leu 50 55 60 Asp Arg Asp Gln LysSer Val Ser Glu Ser Ser Pro Leu Thr Asn Gly 65 70 75 80 Ser Trp Ile AspHis Arg Pro Ile Ala Glu Asp Pro Met Gln Trp Val 85 90 95 Phe Asn Pro ThrSer Leu Ser Ser Ser Ser Ser Ser Tyr Gln Ser Leu 100 105 110 Asp Asn LysGlu Lys Lys His Phe Lys Ser Gly Asn Phe Ser Ser Leu 115 120 125 Pro HisPhe Thr Asp Leu Thr Thr Tyr Met Ile Lys Asn Val Ile Asn 130 135 140 PheGly Lys Thr Leu Thr Met Phe Arg Ala Leu Val Met Glu Asp Gln 145 150 155160 Ile Ser Leu Leu Lys Gly Ala Thr Phe Glu Ile Ile Leu Ile His Phe 165170 175 Asn Met Phe Phe Asn Glu Val Thr Gly Ile Trp Glu Cys Gly Pro Leu180 185 190 Gln Tyr Cys Met Asp Asp Ala Phe Arg Ala Gly Phe Gln His HisLeu 195 200 205 Leu Asp Pro Met Met Asn Phe His Tyr Thr Leu Arg Lys LeuArg Leu 210 215 220 His Glu Glu Glu Tyr Val Leu Met Gln Ala Leu Ser LeuPhe Ser Pro 225 230 235 240 Asp Arg Pro Gly Val Thr Asp His Lys Val IleAsp Arg Asn Gln Glu 245 250 255 Thr Leu Ala Leu Thr Leu Lys Thr Tyr IleGlu Ala Lys Arg Asn Gly 260 265 270 Pro Glu Lys His Leu Leu Phe Pro LysIle Met Gly Cys Leu Thr Glu 275 280 285 Met Arg Ser Met Asn Glu Glu TyrThr Lys Gln Val Leu Lys Ile Gln 290 295 300 Asp Met Gln Pro Glu Val SerPro Leu Trp Leu Glu Ile Ile Ser Lys 305 310 315 320 Asp Thr

What is claimed is:
 1. An isolated pregnane X nuclear receptorpolypeptide comprising: (a) an amino acid sequence of SEQ ID NO: 2, 4, 6or 8; (b) a variant of the amino acid sequence as defined in (a) whichmodulates P450 3A4 levels or activity; or (c) a fragment of (a) or (b)which modulates P450 3A4 levels or activity.
 2. A polypeptide accordingto claim 1 wherein the variant (b) has at least 80% identity to theamino acid sequence of SEQ ID NO: 2, 4, 6 or
 8. 3. A polynucleotideencoding a polypeptide according to claim
 1. 4. A polynucleotideaccording to claim 3 which is a cDNA sequence.
 5. A polynucleotideencoding a pregnane X receptor polypeptide which modulates P450 3A4levels or activity, said polynucleotide comprising: (a) a nucleic acidsequence of SEQ ID NO: 1, 3, 5 or 7; (b) a nucleic acid sequence whichhybridizes under stringent conditions to the nucleic acid sequence asdefined in (a); (c) a nucleic acid sequence that is degenerate as aresult of the genetic code to the nucleic acid sequence as defined in(a) or (b); or (d) a nucleic acid sequence having at least 60% identityto the nucleic acid sequence as defined in (a), (b) or (c).
 6. Thepolynucleotide of claim 3 wherein the polynucleotide encodes amino acids106 to 434 set forth in SEQ ID NO:
 4. 7. The polynucleotide of claim 3wherein the polynucleotide encodes amino acids 41 to 105 set forth inSEQ ID NO:4.
 8. The polynucleotide of claim 3 wherein the polynucleotideencodes amino acids 1 to 40 set forth in SEQ ID NO:4.
 9. A fusionprotein comprising: (a) a DNA binding or ligand binding domain of thepregnane X receptor of claim 1; and (b) a non-pregnane Xreceptor-derived amino acid sequence.
 10. An isolated polynucleotideencoding the fusion protein of claim
 9. 11. An expression vectorcomprising a polynucleotide according to any one of claims 3 to 8 or 10.12. A host cell comprising an expression vector according to claim 11.13. An antibody specific for a polypeptide according to claim
 1. 14. Amethod for the identification of a compound that modulates pregnane Xreceptor activity and/or expression, said method comprising: (a)contacting a test compound with a canine, porcine, primate or Zebrafishpregnane X recpetor polypeptide or polynucleotide; and (b) determiningan effect of the test compound on the activity and/or expression of saidpolypeptide or polynucleotide.
 15. A method according to claim 14wherein the polypeptide is expressed in a cell.
 16. A substance whichmodulates pregnane X receptor activity and which is identifiable by amethod according to claim 14 or
 15. 17. A non-human transgenic animalexpressing a PXR polypeptide of claim 1 or a mutant thereof.
 18. Amethod for selecting a preclinical animal model which is predictive ofaffects of a test compound on P450 3A4 in humans comprising comparing invitro activation of human PXR in the presence of the test compound within vitro activation of PXRs from preclinical animal models in thepresence of the test compound, wherein a PXR from a preclinical animalmodel exhibiting similar in vitro activation to the human PXR isindicative of the preclinical animal model being predictive of theaffects of the test compound on P450 3A4 in humans.
 19. The method ofclaim 18 wherein the pregnane X receptor from the preclinical model is acanine, porcine, primate or Zebrafish pregnane X receptor.